title: The contribution of heavy metals in cigarette smoke condensate to malignant transformation of breast epithelial cells and in vivo initiation of neoplasia through induction of a PI3K-AKT-NFκB cascade.
abstract: Cigarette smoking is a crucial factor in the development and progression of multiple cancers including breast. Here, we report that repeated exposure to a fixed, low dose of cigarette smoke condensate (CSC) prepared from Indian cigarettes is capable of transforming normal breast epithelial cells, MCF-10A, and delineate the biochemical basis for cellular transformation. CSC transformed cells (MCF-10A-Tr) were capable of anchorage-independent growth, and their anchorage dependent growth and colony forming ability were higher compared to the non-transformed MCF-10A cells. Increased expression of biomarkers representative of oncogenic transformation (NRP-1, Nectin-4), and anti-apoptotic markers (PI3K, AKT, NFκB) were also noted in the MCF-10A-Tr cells. Short tandem repeat (STR) profiling of MCF-10A and MCF-10A-Tr cells revealed that transformed cells acquired allelic variation during transformation, and had become genetically distinct. MCF-10A-Tr cells formed solid tumors when implanted into the mammary fat pads of Balb/c mice. Data revealed that CSC contained approximately 1.011μg Cd per cigarette equivalent, and Cd (0.0003μg Cd/1×10(7) cells) was also detected in the lysates from MCF-10A cells treated with 25μg/mL CSC. In similar manner to CSC, CdCl2 treatment in MCF-10A cells caused anchorage independent colony growth, higher expression of oncogenic proteins and increased PI3K-AKT-NFκB protein expression. An increase in the expression of PI3K-AKT-NFκB was also noted in the mice xenografts. Interestingly, it was noted that CSC and CdCl2 treatment in MCF-10A cells increased ROS. Collectively, results suggest that heavy metals present in cigarettes of Indian origin may substantially contribute to tumorigenesis by inducing intercellular ROS accumulation and increased expression of PI3K, AKT and NFκB proteins.
authors.affiliation: Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024, India.
authors.forename: Purusottam
authors.initials: P
authors.lastname: Mohapatra
authors.affiliation: Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024, India.
authors.forename: Ranjan
authors.initials: R
authors.lastname: Preet
authors.affiliation: Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024, India.
authors.forename: Dipon
authors.initials: D
authors.lastname: Das
authors.affiliation: Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024, India.
authors.forename: Shakti Ranjan
authors.initials: SR
authors.lastname: Satapathy
authors.affiliation: Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024, India; Department of Infection Biology, Institute of Life Science, Nalco Square, Bhubaneswar, Orissa 751021, India.
authors.forename: Sumit
authors.initials: S
authors.lastname: Siddharth
authors.affiliation: Department of Infection Biology, Institute of Life Science, Nalco Square, Bhubaneswar, Orissa 751021, India.
authors.forename: Tathagata
authors.initials: T
authors.lastname: Choudhuri
authors.affiliation: Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Wyatt
authors.affiliation: Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024, India. Electronic address: cnkundu@gmail.com.
authors.forename: Chanakya Nath
authors.initials: CN
authors.lastname: Kundu
mesh_terms: D000818:Animals; D001940:Breast / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D019216:Metals, Heavy / Q000633:toxicity*; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000096:biosynthesis*; D058539:Phosphatidylinositol 3-Kinase / Q000096:biosynthesis*; D051057:Proto-Oncogene Proteins c-akt / Q000096:biosynthesis*; D012906:Smoke / Q000009:adverse effects*; D012907:Smoking / Q000009:adverse effects; D023041:Xenograft Model Antitumor Assays / Q000379:methods
chemical_list: D019216:Metals, Heavy; D016328:NF-kappa B; D012906:Smoke; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt
keywords: 4′, 6-diamidino-2-phenylindole; 7,12-dimethyl benz [a] anthracene; AAS; BP; Breast cancer; CSC; Cigarette smoke condensate; DAPI; DHE; DMBA; DMSO; FITC; H(2)O(2); Heavy metals; MCF-10A; MCF-10A transformed cells; MCF-10A-Tr; MTT; PBS; ROS; SCSC; STR; Transformation; [3-(4,5-dimethylthiazol 2yl-)-2, 5-diphenyl tetrazolium bromide]; atomic absorption spectrometry; benzo [a] pyrene; cigarette smoke condensate; dihydroethidium; dimethyl sulphoxide; fluorescein isothiocyanate; hydrogen peroxide; phosphate buffered saline; reactive oxygen species; short tandem repeat; standard cigarette smoke condensate